Specific coupling reaction measuring method

ABSTRACT

A specific coupling reaction measuring method of this invention for measuring a content of a subject substance in a sample includes the steps of (a) constructing a reaction system including the sample and a magnetic particle on which a specific coupling substance for specifically coupling with the subject substance is immobilized; (b) measuring an optical characteristic of the reaction system; and (c) removing, from the reaction system, an agglutination complex including the subject substance, the specific coupling substance and the magnetic particle by utilizing magnetic force.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 10/704,802,filed Nov. 12, 2003, now U.S. Pat. No. 7,105,359, which claims priorityof Japanese Patent application No. 2002-328056, filed Dec. 11, 2002, thecontents of which are herewith incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a specific coupling reaction measuringmethod for measuring the content of a substance to be measured(hereinafter referred to as a subject substance) that may be included ina sample, and a reagent kit and a specific coupling reaction measuringapparatus for use in the specific coupling reaction measuring method.

In the field of medical care, the content of a protein peculiar to eachdisease present in a human body fluid is generally measured fordiagnosis of various diseases or for examination of the progressingcondition of a disease. In the measurement of the content of a protein,a specific coupling reaction measuring method utilizing a reactionbetween an antigen and an antibody for specifically recognizing a targetprotein as an antigen (namely, an antigen-antibody reaction) is widelyused. Currently, a variety of specific coupling reaction measuringmethods respectively based on various principles have been developed.

Among these various specific coupling reaction measuring methods,well-known measuring methods are immune nephelometry (hereinafterreferred to simply as the nephelometry), immune turbidimetry(hereinafter referred to simply as the turbidimetry) and a slideagglutination in all of which agglutination of an antigen-antibodycomplex generated through an antigen-antibody reaction (hereinafterreferred to as the agglutination complex) is detected in order tomeasure the content of a subject substance in a sample. In theantigen-antibody reaction, turbidity is caused in the reaction systemdue to the generation of the agglutination complex. The extent of theturbidity caused in the reaction system due to the generation of theagglutination complex depends upon the quantity of the antigen and thequantity of the antibody. On the basis of this, the extent of theturbidity caused in the reaction system is optically measured in thenephelometry and the turbidimetry, so as to calculate the quantity ofthe antigen or the antibody based on the optically measured value.Specifically, the agglutination complex is measured on the basis ofchange in the quantity of light scattered in the reaction systemaccording to the nephelometry and is measured on the basis of change inthe quantity of transmitted light reduced through the scattering in thereaction system according to the turbidimetry. In general, the samereaction system can be used in the measurement by these two methods. Inother words, a reaction system that can be dealt with by one of thesemethods can be dealt with by the other method. In the slideagglutination, turbidity or aggregate caused in a reaction system due togeneration of an agglutination complex is visually measured on a slideglass or the like. Also in the slide agglutination, the same reactionsystem as that usable in the nephelometry and the turbidimetry can beused. In these three measuring methods, the measurement is performed byusing a reaction system in which an antigen and an antibody arehomogeneously dispersed, and hence these methods are known genericallyas an “specific coupling reaction measuring method for a homogeneoussystem”.

Also, in the field of the medical care, the contents of several kinds ofsubject substances are measured for diagnosis of various diseases or forexamination of the progressing condition of a disease. For example, in aclinical examination, the contents of all proteins in a humane urine aremeasured as an index of the filtering function of a kidney, or thecontent of erythrocyte (hemoglobin) in a humane urine is measured as anindex of inflammation, calculosis or a tumor of a urinary tract such asacute glomerulonephritis, IgA nephropathy, nephrophthisis, renalinfarction, interstitial nephritis, cystitis, urethritis or prostatitis(for example, see Non-Patent Document 1 below). Furthermore, PatentDocument 2 below discloses, as a specific coupling reaction measuringmethod for a homogeneous system for measuring several kinds of subjectsubstances, a method in which different reaction vessels are preparedfor respective subject substances for individually measuring the subjectsubstances.

It should be noted that methods of measuring the content of a subjectsubstance in a sample by using a reaction of the subject substanceincluded in the sample to a specific coupling substance for specificallycoupling with the subject substance is referred to as “specific couplingreaction measuring method” in the present description. Theaforementioned immune reaction measuring methods are also examples ofthe specific coupling reaction measuring methods.

Patent Document 1: Japanese Laid-Open Patent Publication No. 5-2024

Non-Patent Document 1: “Summary of clinical examinations”, written byIzumi Kanai, edited by Masamitsu Kanai, revised edition 31, published byKanahara Shuppan Kabushiki Kaisha, 1998, p. 156 and pp. 179-180

However, in any of the aforementioned conventional methods, formeasuring a plurality of kinds of subject substances, it is necessary toprepare reaction vessels in the same number as the number of kinds ofsubject substances, and hence a large quantity of sample is necessary.

SUMMARY OF THE INVENTION

The present invention was devised in consideration of the aforementionedconventional situations, and an object of the invention is providing aspecific coupling reaction measuring method in which a plurality ofsubject substances can be measured within one reaction vessel, and areagent kit and a specific coupling reaction measuring apparatus for usein the specific coupling reaction measuring method.

The specific coupling reaction measuring method of this invention formeasuring a content of a subject substance in a sample, includes thesteps of (a) constructing a reaction system including the sample and amagnetic particle on which a specific coupling substance forspecifically coupling with the subject substance is immobilized; (b)measuring an optical characteristic of the reaction system; and (c)removing, from the reaction system, an agglutination complex includingthe subject substance, the specific coupling substance and the magneticparticle by utilizing magnetic force.

In this method, excessive magnetic particle present in the reactionsystem and the agglutination complex including the subject substance,the specific coupling substance and the magnetic particle are removedfrom the reaction system by utilizing the magnetic force in the step (c)after the measurement. Particularly, since the magnetic force is used inthis invention, the reaction system is not chemically affected at all.Therefore, the reaction system obtained after the measurement can beused for measuring another component of the sample by any of variousmeasuring methods.

Also, in the conventional technique to measure two kinds of subjectsubstances, it is necessary to prepare reaction vessels in the samenumber as the number of kinds of subject substances. In contrast,according to this invention, merely one reaction vessel in which areaction system can be constructed can be used for measuring two kindsof subject substances. Accordingly, the quantity of a sample necessaryfor the measurement can be very small in this embodiment. As a result,in the field of, for example, medical care, the quantity of a sample tobe drawn from a patient can be reduced, so as to lighten a burden of thepatient.

In one aspect, the optical characteristic may be the intensity ofscattered light or the quantity of transmitted light.

In one aspect, the magnetic particle preferably has a diameter ofapproximately 0.05 through 2 μm.

In another aspect, the magnetic particle remaining in the reactionsystem is removed from the reaction system in the step (c).

Another specific coupling reaction measuring method of this inventionfor measuring contents of a first subject substance and a second subjectsubstance in a sample, includes the steps of (a) constructing a reactionsystem including the sample and a magnetic particle on which a firstspecific coupling substance for specifically coupling with the firstsubject substance is immobilized; (b) measuring an opticalcharacteristic of the reaction system after the step (a); (c) collectingan agglutination complex including the first subject substance, thefirst specific coupling substance and the magnetic particle by utilizingmagnetic force; (d) adding, to the reaction system, a second specificcoupling substance for specifically coupling with the second subjectsubstance; and (e) measuring an optical characteristic of the reactionsystem after the step (d).

In this method, in measuring the second subject substance, change in theoptical characteristic of the reaction system caused by theagglutination complex including the subject substance, the specificcoupling substance and the magnetic particle is removed by utilizing themagnetic force. Particularly, since the magnetic force is used, thereaction system is not chemically affected at all. Therefore, thisspecific coupling reaction measuring method can attain high reliabilityalso in a measured value of the second subject substance.

Also, in the conventional technique to measure two kinds of subjectsubstances, it is necessary to prepare reaction vessels in the samenumber as the number of kinds of subject substances. In contrast,according to this invention, merely one reaction vessel in which areaction system can be constructed can be used for measuring two or morekinds of subject substances. Accordingly, the quantity of a samplenecessary for the measurement can be very small in this embodiment. As aresult, in the field of, for example, medical care, the quantity of asample to be drawn from a patient can be reduced, so as to lighten aburden of the patient.

In one aspect, the reaction system preferably includes 2 through 6 wt %of polyethylene glycol in the step (d).

In one aspect, the second specific coupling substance is preferablycomposed of an antigen or antibody for specifically coupling with thesecond subject substance and a nonmagnetic particle on which the antigenor antibody is immobilized.

In one aspect, the magnetic particle preferably has a diameter ofapproximately 0.05 through 2 μm and the nonmagnetic particle preferablyhas a diameter of approximately 0.05 through 2 μm.

In one aspect, a combination of the first subject substance and thesecond subject substance may be a combination of human hemoglobin andhuman albumin.

In another aspect, the magnetic particle remaining in the reactionsystem is collected in the step (c).

Still another specific coupling reaction measuring method of thisinvention for measuring contents of n kinds (wherein n is an integer of2 or more) of subject substances in a sample, includes the steps of (a)constructing a reaction system including the sample and a magneticparticle on which a specific coupling substance for specificallycoupling with one subject substance out of subject substances notmeasured yet is immobilized; (b) measuring an optical characteristic ofthe reaction system after the step (a); (c) removing, from the reactionsystem, an agglutination complex including the one subject substance,the specific coupling substance and the magnetic particle by utilizingmagnetic force; (d) determining to return to the step (a) when the step(b) has not been repeated (n−1) times; (e) adding, to the reactionsystem, a specific coupling substance for specifically coupling with oneremaining subject substance not measured yet in the reaction system; and(f) measuring an optical characteristic of the reaction system after thestep (e).

In this method, the agglutination complex including the subjectsubstance, the specific coupling substance and the magnetic particle isremoved by utilizing the magnetic force after measuring the opticalcharacteristic. Since the magnetic force is used, the reaction system isnot chemically affected at all. Therefore, even when a plurality ofsubject substances are measured, the influence of a previously formedagglutination complex is substantially eliminated, so that everymeasurement can attain high reliability in a measured value of theoptical characteristic.

In particular, in the conventional technique to measure two or morekinds of subject substances, it is necessary to prepare reaction vesselsin the same number as the number of kinds of subject substances. Incontrast, according to this embodiment, merely one reaction vessel inwhich a reaction system can be constructed can be used for measuring twoor more kinds of subject substances. Accordingly, the quantity of asample necessary for the measurement can be very small in thisembodiment. As a result, in the field of, for example, medical care, thequantity of a sample to be drawn from a patient can be reduced, so as tolighten a burden of the patient.

In one aspect, the magnetic particle remaining the reaction system isremoved from the reaction system in the step (c).

Still another specific coupling reaction measuring method of thisinvention for measuring contents of n kinds (wherein n is an integer of2 or more) of subject substances in a sample, includes the steps of (a)constructing a reaction system including the sample and a specificcoupling substance for specifically coupling with one subject substanceout of subject substances not measured yet; (b) measuring an opticalcharacteristic of the reaction system after the step (a); (c) adding, tothe reaction system, a magnetic complex including a magnetic particleand a substance capable of coupling with an agglutination complexincluding the one subject substance and the specific coupling substance;(d) removing, by utilizing magnetic force, the agglutination complexcoupled with the magnetic complex; (e) determining, after the step (d),to return to the step (a) when the step (b) has not been repeated (n−1)times; (f) adding, to the reaction system, a specific coupling substancefor specifically coupling with one remaining subject substance notmeasured yet in the reaction system; and (g) measuring an opticalcharacteristic of the reaction system after the step (f).

In this method, the agglutination complex including the subjectsubstance and the specific coupling substance is removed by utilizingthe magnetic force after measuring the optical characteristic. Since themagnetic force is used, the reaction system is not chemically affectedat all. Therefore, even when a plurality of subject substances aremeasured, the influence of a previously formed agglutination complex issubstantially eliminated, so that every measurement can attain highreliability in a measured value of the optical characteristic.

In particular, in the conventional technique to measure two or morekinds of subject substances, it is necessary to prepare reaction vesselsin the same number as the number of kinds of subject substances. Incontrast, according to this embodiment, merely one reaction vessel inwhich a reaction system can be constructed can be used for measuring twoor more kinds of subject substances. Accordingly, the quantity of asample necessary for the measurement can be very small in thisembodiment. As a result, in the field of, for example, medical care, thequantity of a sample to be drawn from a patient can be reduced, so as tolighten a burden of the patient.

In one aspect, the magnetic complex not coupled with the agglutinationcomplex is removed from the reaction system in the step (d).

The reagent kit of this invention for measuring contents of a firstsubject substance and a second subject substance in a sample, includes amagnetic particle on which a first specific coupling substance forspecifically coupling with the first subject substance is immobilized;and a second specific coupling substance for specifically coupling withthe second subject substance.

When this reagent kit of the invention is used in a specific couplingreaction measuring method, the contents of two or more kinds of subjectsubstances can be measured with merely one reaction vessel in which areaction system can be constructed. Accordingly, the quantity of asample necessary for the measurement can be very small.

In one aspect, the magnetic particle preferably has a diameter ofapproximately 0.05 through 2 μm.

In one aspect, the second specific coupling substance is preferablycomposed of an antigen or antibody for specifically coupling with thesecond subject substance and a nonmagnetic particle on which the antigenor antibody is immobilized.

Another reagent kit of this invention for measuring contents of n kinds(wherein n is an integer of 2 or more) of subject substances in asample, includes one specific coupling substance for specificallycoupling with one subject substance out of the n kinds of subjectsubstances; and (n−1) kinds of magnetic particles on which specificcoupling substances for respectively specifically coupling with theother (n−1) kinds of subject substances out of the n kinds of subjectsubstances are respectively immobilized.

When this reagent kit of the invention is used in a specific couplingreaction measuring method, the contents of two or more kinds of subjectsubstances can be measured with merely one reaction vessel in which areaction system can be constructed. Accordingly, the quantity of asample necessary for the measurement can be very small.

Still another reagent kit of this invention for measuring contents of nkinds (wherein n is an integer of 2 or more) of subject substances in asample, includes n kinds of specific coupling substances forrespectively specifically coupling with the n kinds of subjectsubstances; and magnetic complexes each including a substance capable ofcoupling with an agglutination complex including a combination of eachof (n−1) kinds of subject substances out of the n kinds of subjectsubstances and each of (n−1) kinds of corresponding specific couplingsubstances, and a magnetic particle on which the substance isimmobilized.

When this reagent kit of the invention is used in a specific couplingreaction measuring method, the contents of two or more kinds of subjectsubstances can be measured with merely one reaction vessel in which areaction system can be constructed. Accordingly, the quantity of asample necessary for the measurement can be very small.

The specific coupling reaction measuring apparatus of this inventionincludes a cell; a light source for emitting light to the cell; aphotodetector for detecting scattered light or transmitted lightreceived from a reaction system constructed in the cell; and removingmeans for removing a magnetic particle from the cell by utilizingmagnetic force when the cell contains the magnetic particle.

This specific coupling reaction measuring apparatus is suitably used forany of the above-described specific coupling reaction measuring methodsof this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of a specific coupling reaction measuring methodaccording to an embodiment of the invention;

FIGS. 2A, 2B and 2C are schematic diagrams of a reaction system inrespective procedures in the specific coupling reaction measuring methodof FIG. 1;

FIG. 3 is a flowchart of a specific coupling reaction measuring methodaccording to another embodiment of the invention;

FIGS. 4A and 4B are schematic diagrams of a reaction system inrespective procedures in the specific coupling reaction measuring methodof FIG. 3;

FIG. 5 is a flowchart of a specific coupling reaction measuring methodaccording to still another embodiment of the invention;

FIG. 6 is a flowchart of a specific coupling reaction measuring methodaccording to still another embodiment of the invention;

FIGS. 7A and 7B are schematic diagrams of a reaction system inrespective procedures in the specific coupling reaction measuring methodof FIG. 6;

FIG. 8 is a schematic diagram of a measuring apparatus according to anembodiment of the invention;

FIG. 9 is a diagram for showing the arrangement relationship in a partof the measuring apparatus of FIG. 8;

FIG. 10 is a schematic diagram of a measuring apparatus according toanother embodiment of the invention;

FIG. 11 is a diagram for showing the arrangement relationship in a partof the measuring apparatus of FIG. 10; and

FIG. 12 is a diagram for showing another arrangement relationship in apart of the measuring apparatus of FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION

Immune reaction measuring methods, which are example of specificcoupling reaction measuring methods, will now be described in thefollowing embodiments with reference to the accompanying drawings. Forsimplification, like reference numerals are used to refer to likeelements commonly used in the respective embodiments.

Embodiment 1

FIG. 1 is a flowchart of a specific coupling reaction measuring methodof this embodiment. FIGS. 2A through 2C are schematic diagrams of areaction system in respective procedures in the specific couplingreaction measuring method of this embodiment.

First, in step St1 of FIG. 1, a reaction system including a sample 1 inwhich the content of a subject substance 2 is to be measured and amagnetic particle 4 on which a specific coupling substance 3 forspecifically coupling with the subject substance 2 is immobilized isconstructed as shown in FIG. 2A. The sample 1 can be, for example, abody fluid such as a blood or a urine itself, or a mixture of such abody fluid and a buffer. In the case where the subject substance 2 is anantigen, the specific coupling substance 3 is an antibody, and in thecase where the subject substance 2 is an antibody, the specific couplingsubstance 3 is an antigen.

In this manner, when the subject substance 2 is included in the sample,an agglutination complex 5 including the subject substance 2, thespecific coupling substance 3 and the magnetic particle 4 is generatedthrough an antigen-antibody reaction between the subject substance 2 andthe specific coupling substance 3 as shown in FIG. 2B. When the subjectsubstance 2 is not included in the sample, the agglutination complex 5resulting from the antigen-antibody reaction between the subjectsubstance 2 and the specific coupling substance 3 is not generated.

Next, in step St2 of FIG. 1, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex 5 has been generated in step St1, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample 1 obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample 1, thebuffer may be used as a reference.

Next, in step St3 of FIG. 1, the agglutination complex 5 including thesubject substance 2, the specific coupling substance 3 and the magneticparticle 4 and excessive magnetic particles not forming anyagglutination complex 5 (not shown) are collected by utilizing magneticforce (that is, specifically, a magnet 6 as shown in FIG. 2C) to beremoved from the reaction system.

In the specific coupling reaction measuring method, the extent of theturbidity resulting from the generation of the agglutination complexdepends upon the quantity of an antigen and the quantity of an antibody.Accordingly, in this embodiment, the optical characteristic of thereaction system is measured, and the content of the subject substance 2can be calculated on the basis of the measured value of the opticalcharacteristic.

Furthermore, in this embodiment, the excessive magnetic particlespresent in the reaction system and the agglutination complex 5 includingthe subject substance 2, the specific coupling substance 3 and themagnetic particle 4 are removed from the reaction system by utilizingthe magnetic force after the measurement. Since the magnetic force isused in this embodiment, the reaction system is not chemically affectedat all. Therefore, the reaction system obtained after the measurementcan be used for measuring another component of the sample 1 by any ofvarious measuring methods.

Also, in the conventional technique to measure two or more kinds ofsubject substances, it is necessary to prepare reaction vessels in thesame number as the number of kinds of subject substances. In contrast,according to this embodiment, merely one reaction vessel in which areaction system can be constructed can be used for measuring two or morekinds of subject substances. Accordingly, the quantity of a samplenecessary for the measurement can be very small in this embodiment. As aresult, in the field of, for example, medical care, the quantity of asample to be drawn from a patient can be reduced, so as to lighten aburden of the patient.

Moreover, the magnetic particle can be collected to be recycled, withthe immobilized antibody or antigen having reactivity, from theagglutination complex 5 and the excessive magnetic particles (not shown)removed from the reaction system in this embodiment by inhibiting theantigen-antibody reaction under an acidic condition of pH 3.0 or less.Alternatively, the antigen or antibody immobilized on the magneticparticle can be completely removed through a treatment with a strongacid or alkali and washing with a surfactant, so that the resultantmagnetic particle can be used for immobilizing another antibody orantigen.

The magnetic particles 4 used in the specific coupling reactionmeasuring method of this embodiment are preferably insoluble in thereaction system. Specific examples of the magnetic particles 4 areferrite particles, ferrite colloid particles and ferrite-containinglatex particles. Such magnetic particles can be prepared by a user. Inparticular, magnetic particles having substantially uniform diameterscan be obtained as follows: Magnetic bacteria somatically includingmagnetic particles of 0.05 through 0.1 μm are cultured to increase thenumber of bacteria; the resultant bacteria are spalled with a Frenchpress or the like; and the magnetic particles of the bacteria arecollected by using a magnet to be separately taken out. The method forobtaining the magnetic particles from the magnetic bacteria is morespecifically described in Japanese Laid-Open Patent Publication No.2000-346843.

The magnetic particle obtained from the magnetic bacterium is coveredwith a lipid bilayer. Therefore, such magnetic particles are welldispersed in an aqueous solution or the like and are suitably used forimmobilizing a protein such as an antibody used as the specific couplingsubstance.

The diameter of the magnetic particle 4 used in this embodiment ispreferably 0.05 through 2 μm because magnetic particles with such adiameter can be easily homogeneously dispersed in an aqueous solutionand can allow the change in the intensity of scattered light or thequantity of transmitted light to be detected for determining theagglutination complex generated through the antigen-antibody reaction.

The magnet 6 is used for generating the magnetic force to collect themagnetic particles in this embodiment, and the magnet 6 is morespecifically a permanent magnet or an electromagnet.

The reaction system used in the specific coupling reaction measuringmethod of this embodiment may additionally include an arbitrary knowncomponent in accordance with the application. For example, in order toreduce nonspecific agglutination through autoagglutinin of the subjectsubstance 2 and the specific coupling substance 3, a surfactant such asTween 20, octyl glucoside, sodium lauryl sulfate (SDS), sucrosemonolaurate or CHAPS may be added to the reaction system of thisembodiment. The content of the surfactant in the reaction system ispreferably 0.3 wt % or less and more preferably 0.1 wt % or less becausethe surfactant of such a content minimally affects the antigen-antibodyreaction.

In the specific coupling reaction measuring method of this embodiment,as the optical characteristic measured in step St2, change in theintensity of scattered light may be measured (as in the nephelometry) orchange in the quantity of transmitted light may be measured (as in theturbidimetry).

The subject substance of this embodiment is not particularly specifiedbut may be any substance that can be measured by utilizing anantigen-antibody reaction. Examples of the subject substance areprotein, nucleic acid, lipid, bacteria, virus and hapten. In particular,the specific coupling reaction measuring method of this embodiment issuitably used for measuring a protein that is conventionally measured byutilizing an antigen-antibody reaction in the clinical examination.Examples of the protein are hormones such as LH (luteinzing hormone),FSH (follicle-stimulating hormone) and hCG (human chorionicgonadotropin), various immunoglobulin classes and sub-classes, acomponent of complement, markers of various infectious diseases, CRP,albumin, hemoglobin, rheumatoid factors and blood group antigens.

The antibody used in this embodiment is not particularly specified asfar as it can produce an agglutination complex together with an antigenby specifically coupling with the antigen. Examples of the antibody areantibodies of any antibody class of IgG, IgM, IgE, IgA or IgD, and amixture of any of these antibodies. Also, the antibody may be apolyclonal antibody or a monoclonal antibody, or a mixture of them.Among these antibodies, IgG antibodies are preferred because they areless nonspecifically reacted and are comparatively easily commerciallyavailable and hence are easily obtained. Also, the kind of originalanimal of the antibody is not particularly specified, and antibodiesderived from a rabbit, a goat and a mouse are preferred because they arecomparatively easily obtained and widely used.

Although an immune reaction measuring method utilizing anantigen-antibody reaction is described as a specific coupling reactionmeasuring method in this embodiment, the measurement can be performed bygenerating an agglutination complex by using another reaction forcausing specific coupling other than the antigen-antibody reaction. Whenanother reaction for causing specific coupling other than theantigen-antibody reaction is used, a combination of a subject substanceand a specific coupling substance can be, for example, a combination ofa ligand and a receptor or a combination of a single-stranded DNA(subject substance) and various DNA fragments (specific couplingsubstance) having complementary sequences of the single-stranded DNA.

An example of the combination of a ligand and a receptor is acombination of a molecule working as a ligand and an allosteric proteinhaving a plurality of coupling sites with the molecule. In the casewhere the molecule working as a ligand has merely one site to be coupledwith an allosteric protein, an agglutination complex including themolecule working as a ligand and the allosteric protein can be generatedby immobilizing the molecule working as a ligand on a magnetic particlewith a site other than that to be coupled with the allosteric protein.

Alternatively, in the case of a single-stranded DNA (subject substance)and various DNA fragments (specific coupling substance) havingcomplementary sequences of the single-stranded DNA, so as to formagglutination complex, the various DNA fragments may be immobilized on amagnetic particle so as to be complementarily capable of coupling withthe single-stranded DNA.

Embodiment 2

In Embodiment 2 of the invention, a specific coupling reaction measuringmethod in which contents of two kinds of subject substances can bemeasured will be described with reference to the accompanying drawings.

FIG. 3 is a flowchart of the specific coupling reaction measuring methodof this embodiment. FIGS. 4A and 4B are schematic diagrams of a reactionsystem in procedures in the specific coupling reaction measuring methodof this embodiment.

First, in step St21 of FIG. 3, a reaction system including a sample inwhich contents of a first subject substance and a second subjectsubstance are to be measured and a magnetic particle on which a firstspecific coupling substance for specifically coupling with the firstsubject substance is immobilized is constructed. The sample can be, forexample, a body fluid such as a blood or a urine itself, or a mixture ofsuch a blood fluid and a buffer. In the case where the first subjectsubstance is an antigen, the first specific coupling substance is anantibody, and in the case where the first subject substance is anantibody, the first specific coupling substance is an antigen.

In this manner, when the first subject substance 2 a is included in thesample, an agglutination complex 5 a including the first subjectsubstance 2 a, the first specific coupling substance 3 a and themagnetic particle 4 a is generated through an antigen-antibody reactionbetween the first subject substance 2 a and the first specific couplingsubstance 3 a as shown in FIG. 4A. When the first subject substance 2 ais not included in the sample, the agglutination complex 5 a resultingfrom the antigen-antibody reaction between the first subject substance 2a and the first specific coupling substance 3 a is not generated.

Next, in step St22 of FIG. 3, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex 5 a has been generated in step St21, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample, the buffermay be used as a reference.

Next, in step St23 of FIG. 3, the agglutination complex 5 a includingthe first subject substance 2 a, the first specific coupling substance 3a and the magnetic particle 4 a and other excessive magnetic particlesnot forming any agglutination complex 5 a are collected by utilizingmagnetic force. At this point, the agglutination complex 5 a and theexcessive magnetic particles may be removed from the reaction system ormay be collected in a part of a reaction vessel in which the reactionsystem is constructed so as not to disturb optical characteristicmeasurement of the reaction system performed in step St25 describedbelow.

Then, in step St24 of FIG. 3, a second specific coupling substance to bespecifically coupled with the second subject substance is added to thereaction system. For example, in the case where the second subjectsubstance is an antigen, the second specific coupling substance is anantibody, and in the case where the second subject substance is anantibody, the second specific coupling substance is an antigen.

In this manner, when the second subject substance is included in thesample, an agglutination complex 4 b including the second subjectsubstance 2 b and the second specific coupling substance 3 b isgenerated through an antigen-antibody reaction between the secondsubject substance 2 b and the second specific coupling substance 3 b asshown in FIG. 4B. Needless to say, when the second subject substance 2 bis not included in the sample, the agglutination complex 4 b resultingfrom the antigen-antibody reaction between the second subject substance2 b and the second specific coupling substance 3 b is not generated.

Next, in step St25 of FIG. 3, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex 4 b has been generated in step St24, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample, the buffermay be used as a reference.

Particularly, since the agglutination complex 5 a and the excessivemagnetic particles not forming any agglutination complex 5 a arecolleted by utilizing the magnetic force in step St23 as shown in FIG.4B, when the optical characteristic of the reaction system is measuredin this step, the change in the intensity of scattered light and thequantity of transmitted light caused by the agglutination complex 4 balone can be measured.

In particular in this embodiment, in measuring the second subjectsubstance 2 b, the excessive magnetic particles 4 a present in thereaction system and the change in the optical characteristic of thereaction system caused by the agglutination complex 5 a including thefirst subject substance 2 a, the first specific coupling substance 3 aand the magnetic particle 4 have been removed from the reaction systemby utilizing the magnetic force. Since the magnetic force is used inthis embodiment, the reaction system is not chemically affected at all.Therefore, the specific coupling reaction measuring method of thisembodiment can attain high reliability in a measured value also inmeasuring the second subject substance 2 b.

In the conventional technique to measure two kinds of subjectsubstances, it is necessary to prepare two reaction vessels according tothe number of kinds of subject substances. In contrast, according tothis embodiment, merely one reaction vessel in which a reaction systemcan be constructed can be used for measuring two kinds of subjectsubstances. Accordingly, the quantity of a sample necessary for themeasurement is very small in this embodiment. As a result, in the fieldof, for example, the medical care, the quantity of a sample to be drawnfrom a patient can be reduced, so as to lighten a burden of the patient.

Moreover, the magnetic particle can be collected to be recycled, withthe immobilized antibody or antigen having reactivity, from theagglutination complex 5 and the excessive magnetic particles (not shown)removed from the reaction system in this embodiment by inhibiting theantigen-antibody reaction under an acidic condition of pH 3.0 or less.Alternatively, the antigen or antibody immobilized on the magneticparticle can be completely removed through a treatment with a strongacid or alkali and washing with a surfactant, so that the resultantmagnetic particle can be used for immobilizing another antibody orantigen.

Next, a reagent kit used in the specific coupling reaction measuringmethod of this embodiment will be described with reference to FIGS. 4Aand 4B.

The reagent kit used in the specific coupling reaction measuring methodof this embodiment includes a magnetic particle 4 a on which a firstspecific coupling substance 3 a for specifically coupling with a firstsubject substance 2 a is immobilized, and a second specific couplingsubstance 3 b for specifically coupling with a second subject substance2 b.

When the first subject substance 2 a is an antigen, the first specificcoupling substance 3 a is an antibody, and when the first subjectsubstance 2 a is an antibody, the first specific coupling substance 3 ais an antigen. Also, when the second subject substance 2 b is anantigen, the second specific coupling substance 3 b is an antibody, andwhen the second subject substance 2 b is an antibody, the secondspecific coupling substance 3 b is an antigen.

When the reagent kit of this embodiment is used in the specific couplingreaction measuring method of this embodiment, the contents of two kindsof subject substances can be measured with merely one reaction vessel inwhich a reaction system can be constructed. Accordingly, when thereagent kit of this embodiment is used, the quantity of a samplenecessary for the measurement can be very small.

In particular, in measuring the second subject substance 2 b, theexcessive magnetic particles 4 a present in the reaction system and thechange in the optical characteristic of the reaction system caused bythe agglutination complex 5 a including the first subject substance 2 a,the first specific coupling substance 3 a and the magnetic particle 4have been removed from the reaction system by utilizing the magneticforce. Since the magnetic force is used in this embodiment, the reactionsystem is not chemically affected at all. Therefore, the specificcoupling reaction measuring method using the reagent kit of thisembodiment can attain high reliability in a measured value also inmeasuring the second subject substance 2 b.

The magnetic particles 4 a used in the specific coupling reactionmeasuring method and the reagent kit of this embodiment are preferablyinsoluble in a reaction system. Specific examples of the magneticparticles 4 a are ferrite particles, ferrite colloid particles andferrite-containing latex particles. Such magnetic particles can beprepared by a user. In particular, magnetic particles havingsubstantially uniform diameters can be obtained as follows: Magneticbacteria somatically including magnetic particles of 0.05 through 0.1 μmare cultured to increase the number of bacteria; the resultant bacteriaare spalled with a French press or the like; and the magnetic particlesof the bacteria are collected by using a magnet to be separately takenout. The method for obtaining the magnetic particles from the magneticbacteria is more specifically described in Japanese Laid-Open PatentPublication No. 2000-346843.

The magnetic particle obtained from the magnetic bacterium is coveredwith a lipid bilayer. Therefore, such magnetic particles are welldispersed in an aqueous solution or the like and are suitably used forimmobilizing a protein such as an antibody used as the specific couplingsubstance.

The diameter of the magnetic particle 4 a used in this embodiment ispreferably 0.05 through 2 μm because magnetic particles with such adiameter can be easily homogeneously dispersed in an aqueous solutionand can allow the change in the intensity of scattered light or thequantity of transmitted light to be detected for determining theagglutination complex generated through the antigen-antibody reaction.

A magnet 6 is used for generating the magnetic force to collect themagnetic particles in this embodiment, and the magnet 6 is morespecifically a permanent magnet or an electromagnet.

In the specific coupling reaction measuring method and the reagent kitof this embodiment, the second specific coupling substance 3 b is, forexample, an antigen or an antibody but is not limited to this. Inparticular, the second specific coupling substance 3 b is preferably anonmagnetic particle on which an antigen or an antibody to beantigen-antibody reactive with the second subject substance 2 b isimmobilized. Thus, in measuring the second subject substance 2 b(namely, in step St25), the range in the change of the opticalcharacteristic is not largely varied from the range in the change of theoptical characteristic attained in measuring the first subject substance2 a (namely, in step St22). Therefore, an apparatus used for themeasurement can be easily adjusted. The nonmagnetic particle ispreferred also because the progress of the antigen-antibody reaction ofthe second subject substance 2 b is thus not affected by the magneticforce at all.

Examples of the nonmagnetic particle are glass particles, graphiteparticles, gold colloid particles and latex particles. Among theseparticles, the latex particles are preferred because they are highlystable and various diameters are easily available, and preferable latexparticles are polystyrene latex particles good at adsorption to aprotein.

The nonmagnetic particle is required to be easily dispersedhomogeneously in a solution and to allow the change in the intensity ofscattered light and the quantity of transmitted light to be detected fordetermining the agglutination complex generated through theantigen-antibody reaction. Accordingly, the diameter of the nonmagneticparticle is preferably 0.05 through 2 μm, and more preferably equivalentto the diameter of the magnetic particle 4 a because the range in thechange of the optical characteristic can be thus equivalent to thatattained in the measurement using the magnetic particle 4 a.

The reaction system used in the specific coupling reaction measuringmethod of this embodiment may additionally include an arbitrary knowncomponent in accordance with the application. For example, in the casewhere the second specific coupling substance 3 b is not a nonmagneticparticle on which an antigen or an antibody is immobilized, polyethyleneglycol may be added to the reaction system in step St24. Also, in orderto add polyethylene glycol to the reaction system in step St24, thepolyethylene glycol may be included in the reagent kit. The content ofthe polyethylene glycol is preferably 2 through 6 wt % and morepreferably 4 wt % with respect to the reaction system. In the case wherethe polyethylene glycol is included in the reaction system in theaforementioned concentration, nonspecific agglutination throughautoagglutinin of the second subject substance 2 b and the secondspecific coupling substance 3 b can be reduced, so as to improve themeasurement sensitivity.

Furthermore, in order to reduce the nonspecific agglutination throughautoagglutinin of the first subject substance 2 a, the first specificcoupling substance 3 a, the second subject substance 2 b and the secondspecific coupling substance 3 b, a surfactant such as Tween 20, octylglucoside, sodium lauryl sulfate (SDS), sucrose monolaurate or CHAPS maybe added to the reaction system in step St22 and step St25 of thisembodiment. The content of the surfactant in the reaction system ispreferably 0.3 wt % or less and more preferably 0.1 wt % or less becausethe surfactant of such a content minimally affects the antigen-antibodyreaction.

In the specific coupling reaction measuring method of this embodiment,as the optical characteristic measured in step St22 and step St25,change in the intensity of scattered light may be measured (as in thenephelometry) or change in the quantity of transmitted light may bemeasured (as in the turbidimetry).

The first subject substance 2 a and the second subject substance 2 b ofthis embodiment are not particularly specified but may be any substancethat can be measured by utilizing an antigen-antibody reaction. Examplesof the subject substance are protein, nucleic acid, lipid, bacteria,virus and hapten. In particular, the specific coupling reactionmeasuring method and the reagent kit of this embodiment are suitablyused for measuring a protein that is conventionally measured byutilizing an antigen-antibody reaction in the clinical examination.Examples of the protein are hormones such as LH (luteinzing hormone),FSH (follicle-stimulating hormone) and hCG (human chorionicgonadotropin), various immunoglobulin classes and sub-classes, acomponent of complement, markers of various infectious diseases, CRP,albumin, hemoglobin, rheumatoid factors and blood group antigens. Forexample, when the first subject substance 2 a and the second subjectsubstance 2 b of this embodiment are human hemoglobin and human albumin,respectively, this embodiment can provide measurement results suitableto initial screening for a kidney disease. This is because themeasurement result of human urinary hemoglobin can be used as an indexof inflammation, calculosis or a tumor of a urinary tract such as acuteglomerulonephritis, IgA nephropathy, nephrophthisis, renal infarction,interstitial nephritis, cystitis, urethritis or prostatitis and themeasurement result of human urinary albumin is easily affected by changein the total quantity of proteins in a urine, so that the function of akidney can be evaluated on the basis of these measurement results.

The antibody used in this embodiment is not particularly specified asfar as it can produce an agglutination complex together with an antigenby specifically coupling with the antigen. Examples of the antibody areantibodies of any antibody class of IgG, IgM, IgE, IgA or IgD, and amixture of any of these antibodies. Also, the antibody may be apolyclonal antibody or a monoclonal antibody, or a mixture of them.Among these antibodies, IgG antibodies are preferred because they areless nonspecifically reacted and are comparatively easily commerciallyavailable and hence are easily obtained. Also, the kind of originalanimal of the antibody is not particularly specified, and antibodiesderived from a rabbit, a goat and a mouse are preferred because they arecomparatively easily obtained and widely used.

Although an immune reaction measuring method utilizing anantigen-antibody reaction is described as a specific coupling reactionmeasuring method in this embodiment, the measurement can be performed bygenerating an agglutination complex by using another reaction forcausing specific coupling other than the antigen-antibody reaction. Whenanother reaction for causing specific coupling other than theantigen-antibody reaction is used, a combination of a subject substanceand a specific coupling substance can be, for example, a combination ofa ligand and a receptor or a combination of a single-stranded DNA(subject substance) and various DNA fragments (specific couplingsubstance) having complementary sequences of the single-stranded DNA.

An example of the combination of a ligand and a receptor is acombination of a molecule working as a ligand and an allosteric proteinhaving a plurality of coupling sites with the molecule. In the casewhere the molecule working as a ligand has merely one site to be coupledwith an allosteric protein, an agglutination complex including themolecule working as a ligand and the allosteric protein can be generatedby immobilizing the molecule working as a ligand on a magnetic particleor a nonmagnetic particle with a site other than that to be coupled withthe allosteric protein.

Alternatively, in the case of a single-stranded DNA (subject substance)and various DNA fragments (specific coupling substance) havingcomplementary sequences of the single-stranded DNA, so as to formagglutination complex, the various DNA fragments may be immobilized on amagnetic particle or a nonmagnetic particle so as to be complementarilycapable of coupling with the single-stranded DNA.

Embodiment 3

In Embodiment 3 of the invention, a specific coupling reaction measuringmethod for measuring the contents of a further larger number of kinds ofsubject substances, which results from expansion of the specificcoupling reaction measuring method of Embodiment 2, will be describedwith reference to the accompanying drawing.

FIG. 5 is a flowchart of the specific coupling reaction measuring methodof this embodiment.

First, in step St31 of FIG. 5, a reaction system including a sample inwhich contents of n kinds (wherein n is an integer of 2 or more) ofsubject substances are to be measured and a magnetic particle on which aspecific coupling substance for specifically coupling with one of thesubject substances is immobilized is constructed. The sample can be, forexample, a body fluid such as a blood or a urine itself, or a mixture ofsuch a body fluid and a buffer. In the case where the subject substanceis an antigen, the specific coupling substance is an antibody, and inthe case where the subject substance is an antibody, the specificcoupling substance is an antigen.

In this manner, when the subject substance is included in the sample, anagglutination complex including the subject substance, the specificcoupling substance and the magnetic particle, which is equivalent to theagglutination complex 5 a shown in FIG. 4A of Embodiment 2, is generatedthrough an antigen-antibody reaction between the subject substance andthe specific coupling substance. When the subject substance is notincluded in the sample, the agglutination complex resulting from theantigen-antibody reaction between the subject substance and the specificcoupling substance is not generated.

Next, in step St32 of FIG. 5, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex has been generated in step St31, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample, the buffermay be used as a reference.

Next, in step St33 of FIG. 5, the agglutination complex including thesubject substance, the specific coupling substance and the magneticparticle and other excessive magnetic particles not forming anyagglutination complex are removed from the reaction system by utilizingmagnetic force.

Then, in step St34 of FIG. 5, it is determined whether or not step St32has been repeated (n−1) times. When it is determined that step St32 hasbeen repeated (n−1 ) times, the flow proceeds to step St35. When it isdetermined that step St32 has not been repeated (n−1) times, the flowreturns to step St31. In other words, the procedures in steps St31through St33 are repeatedly performed until step St32 is repeated (n−1)times. Through these repeated procedures, among the n kinds of subjectsubstances, (n−1) kinds of subject substances are removed from thereaction system.

Next, in step St35 of FIG. 5, a specific coupling substance to bespecifically coupled with one remaining subject substance not measuredyet in the reaction system is added to the reaction system. At thispoint, for example, in the case where the subject substance is anantigen, the specific coupling substance is an antibody, and in the casewhere the subject substance is an antibody, the specific couplingsubstance is an antigen.

In this manner, when the subject substance is included in the sample, anagglutination complex including the subject substance and the specificcoupling substance, which is equivalent to the agglutination complex 4 bshown in FIG. 4B of Embodiment 2, is generated through anantigen-antibody reaction between the subject substance and the specificcoupling substance. Needless to say, when the subject substance is notincluded in the sample, the agglutination complex resulting from theantigen-antibody reaction between the subject substance and the specificcoupling substance is not generated.

Next, in step St36 of FIG. 5, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex has been generated in step St35, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample, the buffermay be used as a reference.

Particularly, since the agglutination complex and the excessive magneticparticles not forming any agglutination complex are colleted byutilizing the magnetic force in step St33, when the opticalcharacteristic of the reaction system is measured in this step, changein the intensity of scattered light and the quantity of transmittedlight caused by the agglutination complex alone can be measured.

In this embodiment, the agglutination complex including the subjectsubstance, the specific coupling substance and the magnetic particle isremoved after the measurement of the optical characteristic by utilizingthe magnetic force. Since the magnetic force is used, the reactionsystem is not chemically affected at all. Therefore, in measuring aplurality of subject substances, the influences of previously generatedagglutination complexes are substantially eliminated, and hence, everymeasurement of the optical characteristic can attain high reliability ina measured value.

In particular in the conventional technique to measure two or more kindsof subject substances, it is necessary to prepare reaction vessels inthe same number as the number of kinds of subject substances. Incontrast, according to this embodiment, merely one reaction vessel inwhich a reaction system can be constructed can be used for measuring twoor more kinds of subject substances. Accordingly, the quantity of asample necessary for the measurement is very small in this embodiment.As a result, in the field of, for example, the medical care, thequantity of a sample to be drawn from a patient can be reduced, so as tolighten a burden of the patient.

Moreover, the magnetic particle can be collected to be recycled, withthe immobilized antibody or antigen having reactivity, from theagglutination complex 5 and the excessive magnetic particles (not shown)removed from the reaction system in this embodiment by inhibiting theantigen-antibody reaction under an acidic condition of pH 3.0 or less.Alternatively, the antigen or antibody immobilized on the magneticparticle can be completely removed through a treatment with a strongacid or alkali and washing with a surfactant, so that the resultantmagnetic particle can be used for immobilizing another antibody orantigen.

Next, a reagent kit used in the specific coupling reaction measuringmethod of this embodiment will be described.

The reagent kit used in the specific coupling reaction measuring methodof this embodiment includes magnetic particles on which specificcoupling substances for respectively specifically coupling with the(n−1) kinds of subject substances are respectively immobilized, and aspecific coupling substance for specifically coupling with remaining onekind of subject substance.

When the reagent kit of this embodiment is used in the specific couplingreaction measuring method of this embodiment, the contents of two ormore kinds of subject substances can be measured with merely onereaction vessel in which a reaction system can be constructed.Accordingly, when the reagent kit of this embodiment is used, thequantity of a sample necessary for the measurement can be very small.

The magnetic particles and a magnet 6 for collecting the magneticparticles used in the specific coupling reaction measuring method andthe reagent kit of this embodiment can be the same as those described inEmbodiment 2.

In the specific coupling reaction measuring method and the reagent kitof this embodiment, the specific coupling substance added in step St35of FIG. 5 is preferably a nonmagnetic particle on which an antigen or anantibody to be antigen-antibody reactive with the one remaining subjectsubstance not measured yet in the reaction system is immobilized. Thus,the range in the change of the optical characteristic attained in stepSt36 is not largely varied from the ranges in the changes of the opticalcharacteristics of the (n−1) kinds of subject substances attained instep St32. Therefore, an apparatus used for the measurement can beeasily adjusted.

The nonmagnetic particle can be the same as that described in Embodiment2. Specifically, the diameter of the nonmagnetic particle is preferably0.05 through 2 μm and more preferably equivalent to the diameter of themagnetic particle.

The reaction system used in the specific coupling reaction measuringmethod of this embodiment may additionally include an arbitrary knowncomponent in accordance with the application. For example, in the casewhere the specific coupling substance is not a nonmagnetic particle onwhich an antigen or an antibody is immobilized, polyethylene glycol maybe added to the reaction system in step St35. Also, in order to addpolyethylene glycol to the reaction system in step St35, thepolyethylene glycol may be included in the reagent kit. The content ofthe polyethylene glycol is preferably 2 through 6 wt % and morepreferably 4 wt % with respect to the reaction system. In the case wherethe polyethylene glycol is included in the reaction system in theaforementioned concentration, nonspecific agglutination throughautoagglutinin of the subject substances and the specific couplingsubstances can be reduced in step St36, so as to improve the measurementsensitivity.

Furthermore, in order to reduce the nonspecific agglutination throughautoagglutinin of the respective subject substances and the respectivespecific coupling substances, a surfactant such as Tween 20, octylglucoside, sodium lauryl sulfate (SDS), sucrose monolaurate or CHAPS maybe added to the reaction system in steps St32 and St36. The content ofthe surfactant in the reaction system is preferably 0.3 wt % or less andmore preferably 0.1 wt % or less because the surfactant of such acontent minimally affects an antigen-antibody reaction.

In the specific coupling reaction measuring method of this embodiment,as the optical characteristic measured in step St32 and step St36,change in the intensity of scattered light may be measured (as in thenephelometry) or change in the quantity of transmitted light may bemeasured (as in the turbidimetry).

The n kinds of subject substances of this embodiment are notparticularly specified but may be any substance that can be measured byutilizing an antigen-antibody reaction. Examples of the subjectsubstance are protein, nucleic acid, lipid, bacteria, virus and hapten.In particular, the specific coupling reaction measuring method and thereagent kit of this embodiment are suitably used for measuring a proteinthat is conventionally measured by utilizing an antigen-antibodyreaction in the clinical examination. Examples of the protein arehormones such as LH (luteinzing hormone), FSH (follicle-stimulatinghormone) and hCG (human chorionic gonadotropin), various immunoglobulinclasses and sub-classes, a component of complement, markers of variousinfectious diseases, CRP, albumin, hemoglobin, rheumatoid factors andblood group antigens.

The antibody used in this embodiment is not particularly specified asfar as it can produce an agglutination complex together with an antigenby specifically coupling with the antigen. Examples of the antibody areantibodies of any antibody class of IgG, IgM, IgE, IgA or IgD, and amixture of any of these antibodies. Also, the antibody may be apolyclonal antibody or a monoclonal antibody, or a mixture of them.Among these antibodies, IgG antibodies are preferred because they areless nonspecifically reacted and are comparatively easily commerciallyavailable and hence are easily obtained. Also, the kind of originalanimal of the antibody is not particularly specified, and antibodiesderived from a rabbit, a goat and a mouse are preferred because they arecomparatively easily obtained and widely used.

Although an immune reaction measuring method utilizing anantigen-antibody reaction is described as a specific coupling reactionmeasuring method in this embodiment, the measurement can be performed bygenerating an agglutination complex by using another reaction forcausing specific coupling other than the antigen-antibody reaction. Whenanother reaction for causing specific coupling other than theantigen-antibody reaction is used, a combination of a subject substanceand a specific coupling substance can be, for example, a combination ofa ligand and a receptor or a combination of a single-stranded DNA(subject substance) and various DNA fragments (specific couplingsubstance) having complementary sequences of the single-stranded DNA.

An example of the combination of a ligand and a receptor is acombination of a molecule working as a ligand and an allosteric proteinhaving a plurality of coupling sites with the molecule. In the casewhere the molecule working as a ligand has merely one site to be coupledwith an allosteric protein, an agglutination complex including themolecule working as a ligand and the allosteric protein can be generatedby immobilizing the molecule working as a ligand on a magnetic particleor a nonmagnetic particle with a site other than that to be coupled withthe allosteric protein.

Alternatively, in the case of a single-stranded DNA (subject substance)and various DNA fragments (specific coupling substance) havingcomplementary sequences of the single-stranded DNA, so as to formagglutination complex, the various DNA fragments may be immobilized on amagnetic particle or a nonmagnetic particle so as to be complementarilycapable of coupling with the single-stranded DNA.

Embodiment 4

In Embodiment 4 of the invention, another specific coupling reactionmeasuring method for measuring the contents of a larger number of kindsof subject substances will be described with reference to theaccompanying drawings.

FIG. 6 is a flowchart of the specific coupling reaction measuring methodof this embodiment. FIGS. 7A and 7B are schematic diagrams of a reactionsystem in procedures in the specific coupling reaction measuring methodof this embodiment.

First, in step St41 of FIG. 6, a reaction system including a sample inwhich contents of n kinds (wherein n is an integer of 2 or more) ofsubject substances are to be measured and a specific coupling substancefor specifically coupling with one of the subject substances notmeasured yet is constructed. The sample can be, for example, a bodyfluid such as a blood or a urine itself, or a mixture of such a bodyfluid and a buffer. In the case where the subject substance is anantigen, the specific coupling substance is an antibody, and in the casewhere the subject substance is an antibody, the specific couplingsubstance is an antigen.

In this manner, when the subject substance is included in the sample, anagglutination complex 5 a including the subject substance 2 a and thespecific coupling substance 3 a is generated through an antigen-antibodyreaction between the subject substance 2 a and the specific couplingsubstance 3 a as shown in FIG. 7A. When the subject substance 2 a is notincluded in the sample, the agglutination complex 5 a resulting from theantigen-antibody reaction between the subject substance 2 a and thespecific coupling substance 3 a is not generated.

Next, in step St42 of FIG. 6, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex has been generated in step St41, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. Alternatively, when the reactionsystem is constructed by using the mixture with a buffer as the sample,the buffer may be used as a reference.

Next, in step St43 of FIG. 6, a magnetic complex that includes amagnetic particle and a substance capable of coupling with theagglutination complex including the one subject substance and thespecific coupling substance (for example, an antigen capable of couplingwith an antigen included in the agglutination complex) is added to thereaction system. Thus, as shown in FIG. 7B, the magnetic complex 8including the antibody 7 and the magnetic particle 4 c is coupled withthe agglutination complex 5 c.

Then, in step S44 of FIG. 6, the agglutination complex coupled with themagnetic complex and other excessive magnetic complexes not coupled withany agglutination complex are removed by utilizing magnetic force. Thus,the agglutination complex 5 c is attracted by the magnetic forcetogether with the magnetic complex 8 to be removed, and another subjectsubstance 2 b remains in the reaction system as shown in FIG. 7B.

Then, in step St45 of FIG. 6, it is determined whether or not step St42has been repeated (n−1) times. When it is determined that step St42 hasbeen repeated (n−1) times, the flow proceeds to step St46. When it isdetermined that step St42 has not been repeated (n−1) times, the flowreturns to step St41. In other words, the procedures in steps St4lthrough St44 are repeatedly performed until step St42 is repeated (n−1)times. Through these repeated procedures, among the n kinds of subjectsubstances, (n−1) kinds of subject substances are removed from thereaction system.

Next, in step St46 of FIG. 6, a specific coupling substance to bespecifically coupled with one remaining subject substance not measuredyet in the reaction system is added to the reaction system. At thispoint, for example, in the case where the subject substance is anantigen, the specific coupling substance is an antibody, and in the casewhere the subject substance is an antibody, the specific couplingsubstance is an antigen.

In this manner, when the subject substance is included in the sample, anagglutination complex including the subject substance and the specificcoupling substance, which is equivalent to the agglutination complex 4bshown in FIG. 4B of Embodiment 2, is generated through anantigen-antibody reaction between the subject substance and the specificcoupling substance. Needless to say, when the subject substance is notincluded in the sample, the agglutination complex resulting from theantigen-antibody reaction between the subject substance and the specificcoupling substance is not generated.

Next, in step St46 of FIG. 6, an optical characteristic of the reactionsystem is measured. At this point, in the case where the agglutinationcomplex has been generated in step St45, turbidity is caused in thereaction system, and hence, the intensity of scattered light and thequantity of transmitted light are changed. Accordingly, the extent ofthe turbidity of the reaction system can be estimated by measuring theintensity of scattered light or the quantity of transmitted light. Atthis point, the optical change of the reaction system, namely, thechange in the intensity of scattered light or the quantity oftransmitted light, is preferably measured with reference to that of thesample obtained before the measurement. When the reaction system isconstructed by using the mixture with a buffer as the sample, the buffermay be used as a reference.

Particularly, since the agglutination complex and the magnetic complexare colleted by utilizing the magnetic force in step St44, when theoptical characteristic of the reaction system is measured in this step,change in the intensity of scattered light and the quantity oftransmitted light caused by the agglutination complex alone can bemeasured.

In this embodiment, the agglutination complex including the subjectsubstance and the specific coupling substance are removed after themeasurement of the optical characteristic by utilizing the magneticforce. Since the magnetic force is used, the reaction system is notchemically affected at all. Therefore, in measuring a plurality ofsubject substances, the influences of previously generated agglutinationcomplexes are substantially eliminated, and hence, every measurement ofthe optical characteristic can attain high reliability in a measuredvalue.

In particular in the conventional technique to measure two or more kindsof subject substances, it is necessary to prepare reaction vessels inthe same number as the number of kinds of subject substances. Incontrast, according to this embodiment, merely one reaction vessel inwhich a reaction system can be constructed can be used for measuring twoor more kinds of subject substances. Accordingly, the quantity of asample necessary for the measurement is very small in this embodiment.As a result, in the field of, for example, the medical care, thequantity of a sample to be drawn from a patient can be reduced, so as tolighten a burden of the patient.

Moreover, the magnetic particle can be collected to be recycled, withthe immobilized antibody or antigen having reactivity, from theagglutination complex 5 and the excessive magnetic particles (not shown)removed from the reaction system in this embodiment by inhibiting theantigen-antibody reaction under an acidic condition of pH 3.0 or less.Alternatively, the antigen or antibody immobilized on the magneticparticle can be completely removed through a treatment with a strongacid or alkali and washing with a surfactant, so that the resultantmagnetic particle can be used for immobilizing another antibody orantigen.

Next, a reagent kit used in the specific coupling reaction measuringmethod of this embodiment will be described.

The reagent kit used in the specific coupling reaction measuring methodof this embodiment includes specific coupling substances forrespectively specifically coupling with the n kinds of subjectsubstances, and magnetic complexes respectively including substancesrespectively capable of coupled with agglutination complexes eachincluding each of the (n−1) kinds of subject substances andcorresponding specific coupling substances and magnetic particles onwhich the substances are respectively immobilized.

When the reagent kit of this embodiment is used in the specific couplingreaction measuring method of this embodiment, the contents of two ormore kinds of subject substances can be measured with merely onereaction vessel in which a reaction system can be constructed.Accordingly, when the reagent kit of this embodiment is used, thequantity of a sample necessary for the measurement can be very small.

The magnetic particles and a magnet 6 for collecting the magneticparticles used in the specific coupling reaction measuring method andthe reagent kit of this embodiment can be the same as those described inEmbodiment 2.

As the magnetic complex for specifically coupling with the agglutinationcomplex used in this embodiment can be a magnetic particle on which anantibody for specifically coupling with the antibody forming eachagglutination complex, an antibody specifically coupling with adifferent site from the antibody forming each agglutination complex oran antibody specifically coupled with a specific structure of eachagglutination complex is immobilized.

In the specific coupling reaction measuring method and the reagent kitof this embodiment, the specific coupling substance added in step St46of FIG. 6 is preferably a nonmagnetic particle on which an antigen or anantibody to be antigen-antibody reactive with the one remaining subjectsubstance not measured yet in the reaction system is immobilized. Thus,the range in the change of the optical characteristic attained in stepSt47 is not largely varied from the ranges in the changes of the opticalcharacteristics of the (n−1) kinds of subject substances attained instep St42. Therefore, an apparatus used for the measurement can beeasily adjusted.

The nonmagnetic particle can be the same as that described in Embodiment2. Specifically, the diameter of the nonmagnetic particle is preferably0.05 through 2 μm and more preferably equivalent to the diameter of themagnetic particle.

The reaction system used in the specific coupling reaction measuringmethod of this embodiment may additionally include an arbitrary knowncomponent in accordance with the application. For example, in the casewhere the specific coupling substance is not a nonmagnetic particle onwhich an antigen or an antibody is immobilized, polyethylene glycol maybe added to the reaction system in step St46. Also, in order to addpolyethylene glycol to the reaction system in step St46, thepolyethylene glycol may be included in the reagent kit. The content ofthe polyethylene glycol is preferably 2 through 6 wt % and morepreferably 4 wt % with respect to the reaction system. In the case wherethe polyethylene glycol is included in the reaction system in theaforementioned concentration, nonspecific agglutination throughautoagglutinin of the subject substance and the specific couplingsubstance can be reduced in step St47, so as to improve the measurementsensitivity.

Furthermore, in order to reduce the nonspecific agglutination throughautoagglutinin of the respective subject substances and the respectivespecific coupling substances, a surfactant such as Tween 20, octylglucoside, sodium lauryl sulfate (SDS), sucrose monolaurate or CHAPS maybe added to the reaction system in steps St42 and St47. The content ofthe surfactant in the reaction system is preferably 0.3 wt % or less andmore preferably 0.1 wt % or less because the surfactant of such acontent minimally affects an antigen-antibody reaction.

In the specific coupling reaction measuring method of this embodiment,as the optical characteristic measured in step St42 and step St47,change in the intensity of scattered light may be measured (as in thenephelometry) or change in the quantity of transmitted light may bemeasured (as in the turbidimetry).

The n kinds of subject substances of this embodiment are notparticularly specified but may be any substance that can be measured byutilizing an antigen-antibody reaction. Examples of the subjectsubstance are protein, nucleic acid, lipid, bacteria, virus and hapten.In particular, the specific coupling reaction measuring method and thereagent kit of this embodiment are suitably used for measuring a proteinthat is conventionally measured by utilizing an antigen-antibodyreaction in the clinical examination. Examples of the protein arehormones such as LH (luteinzing hormone), FSH (follicle-stimulatinghormone) and hCG (human chorionic gonadotropin), various immunoglobulinclasses and sub-classes, a component of complement, markers of variousinfectious diseases, CRP, albumin, hemoglobin, rheumatoid factors andblood group antigens.

The antibody used in this embodiment is not particularly specified asfar as it can produce an agglutination complex together with an antigenby specifically coupling with the antigen. Examples of the antibody areantibodies of any antibody class of IgG, IgM, IgE, IgA or IgD, and amixture of any of these antibodies. Also, the antibody may be apolyclonal antibody or a monoclonal antibody, or a mixture of them.Among these antibodies, IgG antibodies are preferred because they areless nonspecifically reacted and are comparatively easily commerciallyavailable and hence are easily obtained. Also, the kind of originalanimal of the antibody is not particularly specified, and antibodiesderived from a rabbit, a goat and a mouse are preferred because they arecomparatively easily obtained and widely used.

Although an immune reaction measuring method utilizing anantigen-antibody reaction is described as a specific coupling reactionmeasuring method in this embodiment, the measurement can be performed bygenerating an agglutination complex by using another reaction forcausing specific coupling other than the antigen-antibody reaction. Whenanother reaction for causing specific coupling other than theantigen-antibody reaction is used, a combination of a subject substanceand a specific coupling substance can be, for example, a combination ofa ligand and a receptor or a combination of a single-stranded DNA(subject substance) and various DNA fragments (specific couplingsubstance) having complementary sequences of the single-stranded DNA.

An example of the combination of a ligand and a receptor is acombination of a molecule working as a ligand and an allosteric proteinhaving a plurality of coupling sites with the molecule. In the casewhere the molecule working as a ligand has merely one site to be coupledwith an allosteric protein, an agglutination complex including themolecule working as a ligand and the allosteric protein can be generatedby immobilizing the molecule working as a ligand on a magnetic particleor a nonmagnetic particle with a site other than that to be coupled withthe allosteric protein.

Alternatively, in the case of a single-stranded DNA (subject substance)and various DNA fragments (specific coupling substance) havingcomplementary sequences of the single-stranded DNA, so as to formagglutination complex, the various DNA fragments may be immobilized on amagnetic particle or a nonmagnetic particle so as to be complementarilycapable of coupling with the single-stranded DNA.

Embodiment 5

In Embodiment 5 of the invention, a measuring apparatus used forpracticing the specific coupling reaction measuring method of any ofEmbodiments 1 through 4 will be described with reference to theaccompanying drawings.

The measuring apparatus 100 will be first described with reference toFIG. 8. FIG. 8 is a schematic diagram of the measuring apparatus 100 ofthis embodiment.

The measuring apparatus 100 of this embodiment includes, as shown inFIG. 8, a light source 101, a cell (reaction vessel) 102, a cell holder103 for holding the cell 102, a photodetector 104 for detecting lightreceived from the cell 102 and a removing section 108.

The light source 101 is disposed so that emitted light can proceed tothe cell 102.

The photodetector 104 detects scattered light or transmitted lightreceived from a reaction system constructed within the cell 102.

The removing section 108 is used for collecting and removing, byutilizing magnetic force, a magnetic particle, a magnetic complex or anagglutination complex from the reaction system in each of Embodiments 1through 4. In the measuring apparatus 100, the removing section 108includes a permanent magnet 105, a cover 106 for protecting thepermanent magnet 105 and an arm 107 on which the permanent magnet 105and the cover 106 are provided.

Now, the operation of the removing section 108 will be described withreference to FIG. 9. FIG. 9 is a diagram for showing the arrangementrelationship between the cell 102 and the removing section 108 in themeasuring apparatus 100 of this embodiment.

As shown in FIG. 9, the arm 107 can be lowered to insert the permanentmagnet 105 and the cover 106 into the cell 102 and can be elevated todraw up the permanent magnet 105 and the cover 106 from the cell 102.Accordingly, the permanent magnet 105 and the cover 106 are introducedinto the reaction system so as to allow the magnetic particle, themagnetic complex or the agglutination complex to be attached onto thesurface of the cover 106. Subsequently, when the turbidity of thereaction system is substantially eliminated, the permanent magnet 105and the cover 106 are drawn up from the cell 102. Thus, the magneticparticle, the magnetic complex or the agglutination complex can becollected and removed from the reaction system. The magnetic particle,the magnetic complex or the agglutination complex attached onto thecover 106 can be mechanically removed from the cover 106. For example,after removing the cover 106 from the permanent magnet 105, thecollected magnetic particle attached onto the cover 106 may be removedby washing, or the cover 106 may be disposable. Alternatively, when anelectromagnet is used instead of the permanent magnet 105, the magneticparticles may be collected and removed by turning off the magnetic fieldgenerated by the electromagnet.

Next, a measuring apparatus 200 will be described with reference to FIG.10. FIG. 10 is a schematic diagram of the measuring apparatus 200 ofthis embodiment.

The measuring apparatus 200 of this embodiment includes, as shown inFIG. 10, a light source 201, a cell (reaction vessel) 202 having anoutlet 202 a, a cell holder 203 for holding the cell 202, aphotodetector 204 for detecting light received from the cell 202 and aremoving section 208.

The light source 201 is disposed so that emitted light can proceed tothe cell 202.

The photodetector 204 detects scattered light or transmitted lightreceived from a reaction system constructed within the cell 202.

The removing section 208 is used for collecting and removing, byutilizing magnetic force, a magnetic particle, a magnetic complex or anagglutination complex from the reaction system in each of Embodiments 1through 4. In the measuring apparatus 200, the removing section 208includes a electromagnet 205, a vessel 206 for storing a substancedischarged through the outlet 202 a of the cell 202 and an on-off valve207 to be connected to the outlet 202 a of the cell 202.

Now, the operation of the removing section 208 will be described withreference to FIG. 11. FIG. 11 is a diagram for showing the arrangementrelationship between the cell 202 and the removing section 208 in themeasuring apparatus 200 of this embodiment.

The valve 207 is connected to the outlet 202 a of the cell 202 as shownin FIG. 11. When electric power is supplied to the electromagnet 205under this condition, a magnetic particle, a magnetic complex or anagglutination complex included in the reaction system within the cell202 is moved through the outlet 202 a and the valve 207 to the vessel206. Subsequently, when the turbidity of the reaction system issubstantially eliminated, the valve 207 is closed and the power supplyto the electromagnet 205 is stopped. Thus, the magnetic particle, themagnetic complex or the agglutination complex can be collected andremoved from the reaction system. The magnetic particle, the magneticcomplex or the agglutination complex stored in the vessel 206 may beremoved by washing, or the vessel 206 may be disposable.

In the measuring apparatus 200, the vessel 206 may be replaced with adischarge pipe connected to the cell 202.

Also, in the measuring apparatus 200, the removing section 208 may bereplaced with a removing section 208′ shown in FIG. 12. As shown in FIG.12, the removing section 208′ includes a permanent magnet 105, an arm107 for holding the permanent magnet 105, a vessel 206 for storing asubstance discharged through the outlet 202 a of the cell 202, and anon-off valve 207 connected to the outlet 202 a of the cell 202.

The arm 107 can elevate and lower the permanent magnet 105 as shown inFIG. 12. Accordingly, with the permanent magnet 105 placed close to thevessel 206, a magnetic particle, a magnetic complex or an agglutinationcomplex included in the reaction system is discharged to the vessel 206.Subsequently, when the turbidity of the reaction system is substantiallyeliminated, the valve 207 is closed and the permanent magnet 105 ismoved away from the vessel 206. Thus, the magnetic particle, themagnetic complex or the agglutination complex can be collected andremoved from the reaction system. The magnetic particle, the magneticcomplex or the agglutination complex stored in the vessel 206 may beremoved by washing or the vessel 206 may be disposable.

In the case where the removing section 108 or 208′ including thepermanent magnet 105 is used, the removing section 108 or 208′ ispreferably operated with the permanent magnet 105 placed away from thecell 202 during the measurement of the optical characteristic of thereaction system, so that the magnetic particle, the magnetic complex orthe agglutination complex contained in the cell 202 can be collected andremoved after completing the measurement.

In the case where the removing section 208 including the electromagnet205 is used, the electromagnet 205 may be placed close to the cell 202.This is because the electromagnet 205 minimally generates magnetic forcewithout electric power supply. Accordingly, the removing section 208 ispreferably operated so as not to generate the magnetic force by notsupplying electric power during the measurement of the opticalcharacteristic of the reaction system and to generate the magnetic forceby supplying the electric power for collecting and removing the magneticparticle, the magnetic complex or the agglutination complex from thecell 202 after completing the measurement. This is because, in eithercase, the influence of the magnetic force on an antigen-antibodyreaction can be minimized in the measurement of the opticalcharacteristic of the reaction system.

At this point, the vessel 206 is preferably filled with a bufferequivalent to the reaction system. Thus, change in the volume of thereaction system within the cell 202 can be reduced. Also, since thechange in the volume of the reaction system within the cell 202 can bethus reduced, in the measurement of the optical characteristic of thereaction system, the volume of an added reagent alone should beconsidered in the measurement. Accordingly, the concentration of asubject substance can be easily estimated on the basis of the measurevalue of the optical characteristic.

EXAMPLE

An example of the invention will now be described in detail. It is notedthat the present invention is not limited to the following example.

In this example, a reagent kit according to Embodiment 2 will bedescribed assuming that the first subject substance is human hemoglobin,that the second subject substance is human albumin, that the firstspecific coupling substance is an anti-human hemoglobin antibody, thatthe second specific coupling substance is an anti-human albuminantibody, and that the second specific coupling substance is notimmobilized on a nonmagnetic particle.

For preparing buffers and the like in this example, purified waterfiltered with Milli-Q SP TOC (manufactured by Millipore) was used. Also,reagents such as a salt and a buffer not particularly described were allavailable from Wako Pure Chemical Industries, Ltd., an extra purereagent was used as polyethylene glycol 6,000 and reagent chemicals wereused as other reagents.

The anti-human hemoglobin antibody used as the first specific couplingsubstance and the anti-human albumin antibody used as the secondspecific coupling substance were prepared as follows:

First, the anti-human albumin antibody was prepared by purifying,through protein A column chromatography, IgG fractions from an antiserumcollected from a rabbit immunized to human albumin (manufactured by WakoPure Chemical Industries, Ltd.). The protein A immobilization gel filledin the column was one manufactured by Amersham Pharmacia Biotech. Anequilibrium buffer used in the purification had a composition of 1.5 Mof glycine and 3.0 M of NaCl with pH 8.9. An elution buffer had acomposition of 0.1 M of citric acid with pH 4.0.

The purification was carried out as follows: After the equilibriumbuffer in the volume five times of the volume of the gel filled in thecolumn was allowed to pass through the column to equilibrate the column,the antiserum including the antibody in the quantity corresponding to 10through 20% of the total couple volume of the column was diluted twofoldwith the equilibrium buffer, and the diluted antiserum was allowed topass through the column so that the antibody included in the antiserumcould be coupled with protein A. Subsequently, the equilibrium bufferwas allowed to pass through the column until a serum component notadsorbed to protein A did not come out of the column, and the column waswashed. Thereafter, the elution buffer was allowed to pass through thecolumn so as to elute the antibody coupled with protein A. The thuseluted fractions of the antibody were placed in a dialysis tube withmolecular cutoff of 10,000 and dialyzed several times with a buffer insubstantially a 100-fold volume with a composition of 0.05 M of3-(N-morpholino)propanesulfonic acid (manufactured by Dojin; hereinafterreferred to as MOPS), 0.15 M of NaCl and 0.04 wt % of NaN₃ with pH 7.4,so as to substitute the buffer component. Subsequently, theconcentration of the antibody was estimated through the absorptiometricanalysis at 280 nm, so as to be set to 3.0 mg/ml by adjusting with thebuffer the same as that used in the dialysis. The resultant solution wasused as an anti-human albumin antibody solution. The concentration ofthe antibody is not limited to that herein described. The thus preparedantibody solution can be stored at room temperature, and is storedpreferably at a lower temperature and more preferably at 4° C. foravoiding modification of the antibody.

The anti-human hemoglobin antibody was prepared by purifying, throughprotein G column chromatography, IgG fractions from an antiserumcollected from a goat immunized to human hemoglobin (manufactured bySIGMA; Cat. No. H-7379) and immobilizing the fractions on magneticparticles. The protein G immobilization gel filled in the column usedfor purifying the IgG fractions was one manufactured by AmershamPharmacia Biotech. An equilibrium buffer used in the purification had acomposition of 0.02 M of Na₂HPO₄-NaH₂PO₄ with pH 7.0. An elution bufferhad a composition of 0.1 M of glycine with pH 2.7. A buffer used in thedialysis had a composition of 0.05 M of MOPS, 0.15 M of NaCl and 0.04 wt% of NaN₃ with pH 7.4. The procedures in the purification by the columnchromatography and in the dialysis for substituting the buffer were thesame as those employed for preparing the anti-human albumin antibody.

Subsequently, the concentration of the antibody was estimated throughthe absorptiometric analysis at 280 nm, so as to be set to 3.0 mg/ml byadjusting with the buffer the same as that used in the dialysis. Theresultant solution was used as an anti-human hemoglobin antibodysolution used for immobilizing the anti-human hemoglobin antibody on themagnetic particles.

The magnetic particles can be prepared or are commercially available. Inthis example, iron-containing polystyrene latex particles with adiameter of 1 through 2 μm, specifically, Polystyrene SuperparamagneticMicrospheres (trade name), 1-2 μ(manufactured by Polysciences; Cat. No.18190) were used. The antibody was immobilized on the magnetic particlesas follows:

First, 0.5 ml of 2.5 wt % magnetic particles was placed in a micro tube,to which 0.5 ml of a buffer with a composition of 0.05 M of MOPS and0.04 wt % of NaN₃ with pH 7.4 was added so as to suspend the magneticparticles. The resultant was centrifuged with a centrifuge (manufacturedby TOMY SEIKO Co. Ltd.; Model No. MRX-150) at 12,000 rpm for 30 minutes.Thus, the magnetic particles were precipitated and the supernatant wasremoved.

Subsequently, 1 ml of a buffer with a composition of 0.05 M of MOPS and0.04 wt % of NaN₃ with pH 7.4 was added to the resultant so as tosuspend the magnetic particles. Then, the suspension was centrifuged at12,000 rpm for 30 minutes, so as to precipitate the magnetic particles,and the supernatant was removed. Such suspension and precipitationthrough centrifugation of the magnetic particles were repeated again.

Furthermore, 0.9 ml of a buffer with a composition of 0.05 M of MOPS and0.04 wt % of NaN₃ with pH 7.4 was added to the resultant, so as tosuspend the magnetic particles. Then, 0.1 ml of the anti-humanhemoglobin antibody solution prepared in the aforementioned manner wasadded to the suspension. The resultant solution was stirred at roomtemperature overnight with a rotator (manufactured by TAITEC; Model No.RT-50). After the stirring, the resultant was centrifuged at 12,000 rpmfor 30 minutes, so as to precipitate the magnetic particles again, andthe supernatant was removed. To the resultant, a buffer with acomposition of 0.05 M of MSP, 1 wt % of casein sodium and 0.04 wt % ofNaN₃ with pH 7.4 was added so as to suspend the magnetic particles, andthe suspension was stirred with a rotator for 30 minutes. After thestirring, the resultant was centrifuged at 12,000 rpm for 30 minutes, soas to precipitate the magnetic particles. Such suspension andprecipitation through centrifugation of the magnetic particles wererepeated three times in total. Thus, the surfaces of the magneticparticles on which the antibody was not immobilized were blocked.

Furthermore, a buffer with a composition of 0.05 M of MOPS, 0.15 M ofNaCl, 0.04 wt % of NaN₃ and 5 vol % of glycerol with pH 7.4 was added tothe resultant, so as to suspend the magnetic particles. It is noted thatthe concentrations of the antibody and the magnetic particles employedfor the immobilization are not limited to those described above. Thethus obtained antibody solution can be stored at room temperature, andis stored preferably at a lower temperature and more preferably at 4° C.for avoiding modification of the antibody.

Also, a buffer with a composition of 0.05 M of MOPS and 0.04 wt % ofNaN₃ with pH 7.4 was prepared as a first buffer to be added to thereaction system for measuring human hemoglobin, and a buffer with acomposition of 0.05 M of MOPS, 8.6 wt % of polyethylene glycol 6,000 and0.04 wt % of NaN₃ with pH 7.4 was prepared as a second buffer to beadded to the reaction system for measuring human albumin. These bufferswere stored at room temperature.

The magnetic particles on which the anti-human hemoglobin antibody (thefirst specific coupling substance) is immobilized, the anti-humanalbumin antibody (the second specific coupling substance) and thebuffers prepared in the aforementioned manner are combined to give thereagent kit to be used in the specific coupling reaction measuringmethod.

The reagent kit is used as follows: First, a sample including twosubject substances (namely, human hemoglobin and human albumin), themagnetic particle on which the anti-human hemoglobin antibody (the firstspecific coupling substance) was immobilized and the first buffer weremixed to construct a reaction system. Next, an optical characteristic ofthe reaction system was measured. After the measurement, anagglutination complex including the human hemoglobin (the first subjectsubstance), the anti-human hemoglobin antibody (the first specificcoupling substance) and the magnetic particle, and other excessivemagnetic particles not forming any agglutination complex were collectedby utilizing magnetic force, so as to eliminate the turbidity of thereaction system.

Subsequently, the anti-human albumin antibody (the second specificcoupling substance) and the second buffer were mixed with the reactionsystem. Thereafter, an optical characteristic of the resultant reactionsystem was measured. In this manner, the concentrations of the subjectsubstances in the sample could be obtained.

Although not described in this example, the anti-human albumin antibody(the second specific coupling substance) may be immobilized on anonmagnetic particle of latex or gold colloid. For immobilizing theantibody on, for example, latex particles, the aforementionedimmobilization method used for the magnetic particles can be directlyemployed.

Although the surfaces of the particles on which the antibody is notimmobilized are blocked with casein sodium in this example, caseinsodium may be replaced with gelatin, skimmed milk or the like. In thiscase, the concentration of gelatin or the like can be the same as thatof casein sodium.

Furthermore, the buffers were prepared with respect to the respectivesubject substances for constructing the reaction systems in thisexample, but one kind of buffer including a water-soluble polymer suchas polyethylene glycol can be used for constructing the respectivereaction systems.

Moreover, the buffer components and the pH of the antibody solutions arenot limited to those described above as far as they do not harmfullyaffect the specific coupling reaction through the coupling between theantigen and the antibody.

Furthermore, in order to take out hemoglobin from erythrocyte, ahemolyzing agent such as potassium chloride may be added to the reactionsystem or a solution including a hemolyzing agent may be included in thereagent kit.

As described so far, according to the specific coupling reactionmeasuring method and the reagent kit of the present invention, two ormore kinds of subject substances can be measured within merely onereaction vessel.

Accordingly, the present invention provides the specific couplingreaction measuring method in which two or more kinds of subjectsubstances can be measured within one reaction vessel and the quantityof a sample necessary for the measurement can be reduced, and thereagent kit and the measuring apparatus used in the specific couplingreaction measuring method.

The specific coupling reaction measuring method, and the reagent kit andthe specific coupling reaction measuring apparatus for use in the methodaccording to the present invention are useful for diagnosis of variousdiseases and examination of the progressing condition of a disease. Whenthe present invention is applied to measure, for example, humanhemoglobin and human albumin in one sample, it is useful for diagnosisof kidney diseases.

1. A specific coupling reaction measuring method for measuring aplurality of subject substances in a sample, comprising the steps of:(a) constructing a reaction system including said sample and a specificcoupling substance for specifically coupling with a first subjectsubstance; (b) measuring an optical characteristic of said reactionsystem; (c) adding, to said reaction system, a magnetic complexincluding a magnetic particle and a substance capable of coupling withan agglutination complex including said first subject substance and saidspecific coupling substance; (d) removing, by utilizing magnetic force,said agglutination complex coupled with said magnetic complex from saidreaction system; (e) adding, to said reaction system, another specificcoupling substance for specifically coupling with another subjectsubstance; and (f) measuring an optical characteristic of said reactionsystem after the step (e).
 2. The specific coupling reaction measuringmethod of claim 1, wherein said optical characteristic is an intensityof scattered light or a quantity of transmitted light.
 3. The specificcoupling reaction measuring method of claim 1, wherein each saidmagnetic particles has a diameter of approximately 0.05 through 2 μm. 4.The specific coupling reaction measuring method of claim 1, wherein aportion of said magnetic particles are included in said agglutinationcomplex and other magnetic particles remain in said reaction system inthe step (a), and said other magnetic particles remaining in saidreaction system are removed from said reaction system in the step (d).5. The specific coupling reaction measuring method of claim 1, whereinsaid reaction system includes 2 through 6 wt % of polyethylene glycol inthe step (e).
 6. The specific coupling reaction measuring method ofclaim 1, wherein said another specific coupling substance is composed ofan antigen or antibody for specifically coupling with said anothersubject substance and a nonmagnetic particle on which said antigen orantibody is immobilized.
 7. The specific coupling reaction measuringmethod of claim 6, wherein said magnetic particle has a diameter ofapproximately 0.05 through 2 μm and said nonmagnetic particle has adiameter of approximately 0.05 through 2 μm.
 8. The specific couplingreaction measuring method of claim 1, wherein a combination of saidfirst subject substance and said another subject substance is acombination of human hemoglobin and human albumin.